Abstract
The isolation of extracellular vesicles (EVs) from blood is of great importance to understand the biological role of circulating EVs and to develop EVs as biomarkers of disease. Due to the concurrent presence of lipoprotein particles, however, blood is one of the most difficult body fluids to isolate EVs from. The aim of this study was to develop a robust method to isolate and characterise EVs from blood with minimal contamination by plasma proteins and lipoprotein particles. Plasma and serum were collected from healthy subjects, and EVs were isolated by size-exclusion chromatography (SEC), with most particles being present in fractions 8–12, while the bulk of the plasma proteins was present in fractions 11–28. Vesicle markers peaked in fractions 7–11; however, the same fractions also contained lipoprotein particles. The purity of EVs was improved by combining a density cushion with SEC to further separate lipoprotein particles from the vesicles, which reduced the contamination of lipoprotein particles by 100-fold. Using this novel isolation procedure, a total of 1187 proteins were identified in plasma EVs by mass spectrometry, of which several proteins are known as EV-associated proteins but have hitherto not been identified in the previous proteomic studies of plasma EVs. This study shows that SEC alone is unable to completely separate plasma EVs from lipoprotein particles. However, combining SEC with a density cushion significantly improved the separation of EVs from lipoproteins and allowed for a detailed analysis of the proteome of plasma EVs, thus making blood a viable source for EV biomarker discovery.
Highlights
A blood sample is minimally invasive and is one of the most commonly used samples for diagnostic purposes [1]
Western blot revealed that apolipoprotein A1 (Apo-A1), a marker for high-density lipoprotein (HDL) and chylomicrons, was detectable in fractions 7–12, showing that lipoprotein particles were co-isolated with extracellular vesicles (EVs) (Fig. 2c)
Important to note that when the degree of contamination is evaluated by measuring lipoproteins such as Apo-A, Apo-B, and Apo-E, proteins such as Apo-E have been reported to be present on exosomes, those released from pigment cells [18], and EVs isolated from plasma can be covered with low-density lipoprotein (LDL) [13], making it difficult to discriminate between lipoprotein particle contaminants and EV-associated lipoproteins
Summary
A blood sample is minimally invasive and is one of the most commonly used samples for diagnostic purposes [1] Diseased cells, such as tumour cells, and injured or stressed tissues release molecules into the bloodstream, and these molecules can be used to monitor the status of different tissues and organs without obtaining an invasive biopsy, and blood samples are, often referred to as a “liquid biopsy” [2]. Cells and molecules that can be analysed in a liquid biopsy include circulating tumour cells, cell-free DNA and RNA, soluble proteins, and extracellular vesicles (EVs) [2]. Human plasma and serum contain a vast array of particles, including EVs, and a dominating pool of lipid particles such as chylomicrons and multiple types of lipoprotein particles and plasma proteins, making blood one of the most difficult body fluids to isolate EVs from
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